Compositions for boron neutron capture therapy and methods thereof

ABSTRACT

Boron neutron capture therapy can utilize X y  B 20  H 17  L where X is an alkali metal, y is 1 to 4, and L is a two electron donor such as NH 3 , and Na 2  B 10  H 9  NCO, among others. These borane salts may be used free or encapsulated in liposomes. Liposomes may have embedded within their bilayers carboranes to increase the amount of delivered  10  B and/or to increase the tumor specificity of the liposome.

FIELD OF THE INVENTION

This invention generally relates to compositions and methods fortreating tumors, and more particularly to compositions and methods fortreating tumors using borane derivatives both free and liposomeencapsulated.

BACKGROUND OF THE INVENTION

Neutron capture therapy is an attractive method for cancer therapy,specifically the treatment of malignant tumors. The generalized reactioninvolves capture of a thermalized neutron (usually from a nuclearreactor with special moderators and ports) by an appropriate nucleushaving a large neutron capture cross-section. The subsequent decay emitsenergetic particles (alpha particles) which can kill nearby tumor cells.Since the energetic and cytotoxic alpha particles travel only about onecell diameter in tissue, preferably one may specify the cell type to bedestroyed by placing the alpha particle precursors only on or within thetumor cells.

Boron-10 (also designated as ¹⁰ B), for example, has such an appropriatenucleus and has particularly advantageous properties for this scheme.The boron-10/thermal neutron capture reaction is as follows (*indicating an unstable intermediate state of the boron nucleus):

    .sup.10 B+.sup.1 n→[.sup.11 B]*→.sup.7 Li(0.87 Mev.)+.sup.4 He(1.52 Mev.)

In order for this therapy to be effective, sufficient ¹⁰ B must belocalized in a tumor to generate the required density of particles. Thislevel has been variously estimated to be approximately 10-50 μg¹⁰ B/gmtumor. Furthermore, the concentration of ¹⁰ B in normal tissue and bloodshould be limited and preferably less than the concentration in thetumor in order to minimize damage to healthy cells and blood vessels. H.Hatanaka (1986) Boron-Neutron Capture Therapy for Tumors; Nishimura Co.,Ltd. p. 1-16.

Large numbers of boron containing compounds have been tested for theirability to satisfy the above criteria. With few exceptions, all havefailed as not enough boron has localized in the tumor and theconcentration in the blood has been too high for effective neutroncapture therapy. Human clinical trials with Na₂ B₁₂ H₁₁ SH in Japan haveshown some promise, but only for a limited group of brain tumors. Id.16-26.

Neutron capture therapy would be greatly expanded in usefulness if ageneralized method for delivering high concentrations of ¹⁰ B to tumorswere available. It would further be useful if more ¹⁰ B collected intumor than in the blood.

Recently it has become possible to deliver drugs and other compoundsselectively to tumors using liposomes of a particular compositionstructure. See European Patent Application No. 87311040.7 published Jun.22, 1988; U.S. Pat. No. 5,019,369 to Presant; and "Liposomes fromBiophysics to Therapeutics", M. J. Ostro, Ed., Marcel Dekker, Inc., NewYork (1987), all of which are incorporated herein by reference.

Incorporation of compounds with higher osmolarity inside the internalspace of liposomes than outside, as is necessary for effective neutroncapture therapy, depends on incorporating the highest concentration of¹⁰ B possible without substantially altering the liposome's favorablebio-distribution characteristics. Thus, the objective of at least 10 μg¹⁰ B per gram of tumor tissue can be met (assuming use of greater than90% ¹⁰ B enriched material).

Na₂ B₂₀ H₁₈ and its hydroxide derivatives are known. See M. F.Hawthorne, R. L. Pilling, and P. M. Garrett, J. Am. Chem. Soc. 87, 4740(1965). It is known to use boron containing polyphosphonates for thetreatment of calcific tumors. See European Patent Application No.82200784.5 published May 1, 1983. Boronated porphyrin compounds for usein neutron capture therapy are also known. See U.S. Pat. No. 4,959,356to Miura, U.S. Pat. No. 5,116,980 to Gabel and U.S. Pat. No. 4,466,952to Hadd.

There is a continuing long felt but unmet need for a method ofselectively delivering therapeutic concentrations of ¹⁰ B to tumors.There is a similar need for ¹⁰ B compositions and delivery vehicleswhich can be used in boron neutron capture therapy.

OBJECTS OF THE INVENTION

It is an object of the invention to provide compositions and methods fordelivering therapeutically useful concentrations of boron containingcompounds to tumors for use in neutron capture tumor therapy.

It is a further object to provide borane and liposome encapsulatedborane compounds that have the properties of retaining concentrations ofsaid borane compounds inside the liposomes without significant breakageof the liposomes.

It is a further object of the invention to provide a method of cancertherapy through use of both free and liposomal encapsulated boranecompounds with the means to deliver at least 10 micrograms ¹⁰ B per gramof tumor tissue to animal and human tumors, while minimizing theconcentration of ¹⁰ B in the blood.

SUMMARY OF THE PREFERRED EMBODIMENTS

The above objectives are fulfilled by the present invention. In oneaspect of the present invention therapeutically effective boranederivatives having two electron donors on the borane cage areencapsulated within the internal aqueous space of liposomes, and theliposomes thereafter administered to a tumor bearing patient. In anotheraspect of the present invention, certain free boranes useful for neutroncapture therapy have been found to have favorable biodistributions.Preferably both free and liposome encapsulated Na₃ B₂₀ H₁₇ NH₃ are usedin these aspects of the invention. In another aspect of the presentinvention, therapeutically effective carborane derivatives are embeddedwithin the liposome bilayer for subsequent administration. The resultingliposomes have heightened tumor selectivity and can be used as anencapsulation vehicle for the previously mentioned borane derivativesand for other drugs. In yet another aspect of the present inventionnovel derivatized boranes are developed for use in boron neutron capturetherapy.

DESCRIPTION OF THE FIGURES

FIG. 1 is a drawing showing liposome membrane constituents and theirstructure.

FIG. 2 shows biodistributions of Na₂ B₁₀ H₁₀ and Na₂ B₁₂ H₁₁ SH inBALB/c mice bearing EMT6 tumors.

FIG. 3 is a proposed reaction of B₁₀ H₉ NCO²⁻ with intracellularprotein.

FIG. 4 shows the biodistributions of Na₂ B₁₀ H₁₀ and Na₂ B₁₀ H₉ NCO inBALB/c mice bearing EMT6 tumors.

FIG. 5 shows a variety of boron agents capable of intracellular bindingwhich are derivatives of B₁₀ H₁₀ ²⁻.

FIG. 6 shows the X-ray crystal structure and ¹¹ B NMR of the tetramethylammonium salt of B₂₀ H₁₇ NH₃ ³⁻.

FIG. 7 shows the biodistribution of liposomal Na₃ B₂₀ H₁₇ NH₃ in BALB/cmice bearing EMT6 tumors.

FIG. 8 shows the intracellular oxidation reaction and the subsequentnucleophilic attack by intracellular proteins.

FIG. 9 shows another biodistribution of liposomal Na₃ B₂₀ H₁₇ NH₃ inBALB/c mice bearing EMT6 tumors.

FIG. 10 shows the biodistribution of free Na₃ B₂₀ H₁₇ NH₃ in BALB/c micebearing EMT6 tumors.

FIG. 11 shows the tumor boron retention of liposomal boranes in BALB/cmice bearing EMT6 tumors.

FIG. 12 shows the development of lipophilic boron species forphospholipid bilayer embedment.

FIG. 13 shows the development of lipophilic boron species forphospholipid bilayer embedment.

FIG. 14 shows the biodistribution of liposomal K[CH₃ (CH₂)₁₅ C₂ B₉ H₁₁ ]in BALB/c mice bearing EMT6 tumors.

FIG. 15 shows the biodistribution of liposomal K[CH₃ (CH₂)₁₅ C₂ B₉ H₁₁ ]in BALB/c mice bearing EMT6 tumors.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following is a description of the preferred embodiments of thepresent invention including the best mode presently contemplated by theinventors.

"Vesicle" refers to a micelie which is in a generally spherical form,often obtained from a lipid which forms a bilayered membrane and isreferred to as a "liposome". Liposomes are microscopic structuresconsisting in part of phospholipids. Methods for forming these liposomesare, by now, well known in the art and any such methods can be employedin the context of the present invention. See, e.g., U.S. Pat. No.4,753,788 to Gamble and U.S. Pat. No. 4,935,171 to Braken. Typically,they are prepared from a phospholipid, for example, distearoylphosphatidylcholine (also known as "DSPC"), and may include othermaterials such as neutral lipids, for example, cholesterol, and alsosurface modifiers such as positively or negatively charged compounds.Phospholipids are composed of two fatty acid chains condensed withglycerol with an additional substitution of a phosphate ester headgroup. By incorporating certain phospholipid molecules, a liposome isobtained which is stable in vivo. It is known that phase transitionpoints are a function of hydrocarbon chain length, see Lanford, TheHydrophobic Effect, 2nd Ed. (1980). Certain phospholipid moleculesexhibit phase transitions at relatively high temperatures (greater than37° C.) and use of these phospholipids in compositions described hereinprovide liposomes with improved stability in vivo.

The stability of the DSPC micelies may be enhanced by the incorporationof cholesterol. Positively charged molecules such as stearylamine oraminomannose or aminomannitol derivatives of cholesterol or negativelycharged molecules such as dialkyl phosphate may also be incorporatedinto the vesicles. [Certain carborane species, as discussed in detailbelow, may also be incorporated into the vesicle's bilayer.]

When phospholipid micelies are introduced into the blood stream, themicelies move to the specific locations of cancerous growth in thepatient's body. To enhance movement of the phospholipid vesicles to thespecific locations, positively charged phospholipid vesicles may firstbe introduced into the patient's blood stream to block the macrophagesor other phagocytic cells in the patient's body. The positively chargedmolecules bound to such phospholipid vesicles may be an aminomannose oraminomannitol derivative of cholesterol. Concurrently or after asuitable period of time such as approximately one (1) hour, otherphospholipid vesicles may be introduced into the patient's blood streamto move to the specific locations in the body. Such phospholipidvesicles may include cholesterol and may be neutral or may be positivelycharged as by the inclusion of a stearylamine or aminomannose oraminomannitol derivative of cholesterol or may be negatively charged asby the inclusion of a dicetyl phosphate.

A wide variety of lipid particles may form delivery vesicles which arecapable of the intact intracellular transport of the encapsulatedcontents. For example, other phospholipid delivery vehicles, such asdisclosed in the Vestar, Inc. patent publication EP0272091 which isincorporated herein by reference, may be employed. These vehicles arecomposed of a single encapsulating phospholipid membrane associated withan amphiphile-associated substrate. However, the lipid particles arepreferably comprised of phospholipids and most preferably are liposomes.

As noted above, either as multilamellar or unilamellar vesicles,liposomes have proven valuable as vehicles for drug delivery in animalsand in humans. Active drugs, including small hydrophilic molecules andpolypeptides, can be trapped in the aqueous core of the liposome, whilehydrophobic substances can be dissolved in the liposome bilayermembrane. The liposome structure can be readily injected and can formthe basis for both sustained release and drug delivery to specific celltypes, or parts of the body. Multilamellars, primarily because they arerelatively large, are usually rapidly taken up by thereticuloendothelial system (the liver and spleen). The inventiontypically utilizes vesicles which remain in the circulatory system forhours and break down after internalization by the target cell. For theserequirements, the tumor treating agents of the present inventionpreferably utilize unilamellars having a diameter of less than 250 nm,and more preferably less than 100 nm.

With reference to FIG. 1, when hydrated, phospholipids form into bilayerstructures with their fatty acid hydrocarbon tails pointed inward andthe polar head groups outward. See K. Shelly, D. A. Feakes, M. F.Hawthorne, P. G. Schmidt, T. A. Krisch, W. F. Bauer, Proc. Natl. Acad.Sci. USA, 1992, 89 9039, which is incorporated herein by reference. Themembrane bilayers in these structures typically encapsulate an aqueousvolume, and form a permeability barrier between the encapsulated volumeand the exterior solution. A hydrated phospholipid suspension, whenagitated, forms multilamellar vesicles (MLV) like tiny onions, withwater separating many bilayers in an onion-like form having diameters of1-10 μm (1000-10,000 nm). Application of a shearing or homogenizingforce such as sonication to an MLV suspension produces small unilamellarvesicles of a size range generally 30-250 nm and preferably 50 to 100 nmin average diameter. Unilamellar is generally meant to include from oneto three, and preferably one, bilayer. The diameter of the liposomeencapsulated borane tumor treating agents of the present invention isrelated to sonication time as well as the method by which the liposomeis prepared. The range of 50 to 100 nm is considered to be preferablefrom the standpoint of maximal circulation time in vivo and greatertumor specificity. In addition, if the liposomes are too large, theliver and the spleen take up too much of the encapsulated borane tumortreating agent. The actual equilibrium diameter is largely determined bythe nature of the phospholipid used and the extent of incorporation ofother lipids such as cholesterol.

Liposomes which have been applied to boron neutron capture therapy cancarry hydrophilic salts of polyhedral borane anions of the presentinvention as solutes in the aqueous internal space of the vesicle. Asdiscussed below, liposomes of the type employed for boron delivery arecapable of excellent localization in a variety of tumors followingintravenous injection.

Other lipids may be combined with phospholipids to produce liposomeshaving particular properties. Specifically, sterols such as cholesterolhelp stabilize the bilayer towards leakage and destruction in bloodplasma. A stable liposome may be obtained by incorporating 5-50%cholesterol by weight of phospholipid into the liposome. Charged lipidsor lipids with specific ligand functions may also be included. Forexample, although the liposome shown in FIG. 1 is comprised of aphospholipid having no net charge on its polar terminus, liposomes withnet negative or positive charges can be prepared by techniques wellknown to those in the art. Small unilamellar vesicles comprised of from3:1 to 1:1 mole ratio and preferably 1:1 mole ratio DSPC and cholesterolare particularly advantageous for delivery of the borane compounds ofthe present invention to tumors.

Boron compounds to be used in neutron capture therapy according to thepresent invention can have two or more atoms of boron per molecule, butpreferably contain at least 10 and more preferably 20 atoms of boron permolecule. The isotopic content of the boron can range from naturalabundance 19.78% ¹⁰ B to greater than 95% ¹⁰ B for a highly enrichedcompound. Natural abundance material is useful for test studies forencapsulation, biodistribution, stability and the like. Highly enrichedmaterial is advantageous for therapy where the maximum practicableconcentration of ¹⁰ B is required.

Boron containing compounds useful for treating tumors according to oneaspect of the present invention are highly water soluble, have small orno charge at physiological pH, are relatively impermeable tophospholipid bilayers of liposomes, and are not toxic or have lowtoxicity to the therapy subject. Examples of such boranes, both free andliposome encapsulated, include those of the formula X_(y) B₂₀ H₁₇ L. Xis selected from the group consisting of alkali metals and tetra alkylammonium. Preferably X is Na, K, Cs or Rb, and alkyl includes methyl,ethyl and other alkyls which do not render the resulting salt insoluble.L is any 2 electron donor and y is 1 to 4. Preferably L is selected fromthe group consisting of --NHR₁ R₂ wherein R₁ and R₂ are the same ordifferent and are selected from the group consisting of hydrogen,benzyl, alkyl and diamine (e.g., ethylene diamine); --SR₁ R₂ wherein R₁and R₂ are the same or different and are selected from the groupconsisting of H or alkyl; --CN; --CO; --NCO; --CH₂ OH; --CO₂ R₁ ;--alkyl; --NHCONHR₁ ; --COOH and --CONHR₁ where R₁ is selected from thegroup consisting of hydrogen, benzyl, alkyl and diamine. Morepreferably, L is selected from the group consisting of --NH₃, --Ph--CH₂--NH₂, --NH₂ CH₂ CH₂ NH₂ and --NH₂ (CH₂)₇ CH₃. Optimally, L is --NH₃.

Liposome encapsulated tumor treating agents of the present inventioninclude a unilamellar liposome where the liposome has at least oneencapsulating bilayer and an internal space defined by the encapsulatingbilayer. A borane compound is encapsulated within the internal space.The borane compound is selected from the group consisting of X_(y) B₂₀H₁₇ L as described above and X_(s) B₁₀ H₉ L, wherein X and L are definedas above and s is 1 or 2. Most preferably, the encapsulated borane isselected from the group consisting of Na_(y) B₂₀ H₁₇ NH₃ where y is 1 or3, and Na₂ B₁₀ H₉ NCO.

A borane concentration inside the liposome of at least 100 mM,preferably 150 mM to 400 mM and more preferably 200 mM to 250 mM,minimizes osmotic pressure while maximizing boron content inside of theliposome, is necessary for a boron compound having at least 10 boronatoms per molecule. Of course, with a lower amount of boron atoms permolecule, a higher concentration is required. The resulting liposomesolution is stable to leakage of the material inside, such that lessthan 10% of the boron material leaks out over a period of 3.5 months.

The present invention extends to methods of performing boron neutroncapture therapy (BNCT) by administering the free boranes and theborane-containing liposomes discussed above and thereafter, subjectingthe patient to a source that emits neutrons. Such a source is described,for example, in U.S. Pat. No. 4,516,535 to Russell, Jr.

Liposome encapsulated borane compounds are prepared by probe sonicationof the dried film preferably composed of equimolar amounts of thephospholipid and cholesterol with the hydrating solution (typically 5ml, 250-300 mM of the borane containing salt). The hydrated lipidsamples can be sonicated using a Sonics & Materials "Vibracell" probesonicator with a microtip operated at the maximum power setting for themicrotip. The solution is maintained at 65° C. under a nitrogenatmosphere and sonicated for about 15-30 minutes. The sample is thenallowed to cool to room temperature to produce small unilamellarvesicles whose average diameter is preferably less than 250 nm and morepreferably 50-100 nm.

The vesicles can be separated from the remaining free borane salt byeluting on a column of Sephadex G-25-80 (medium) with isotonicphosphate-buffered saline or lactose at an osmolarity approximatelyequal to physiological. Liposomal separations can then be diluted withthe appropriate buffer to a lipid concentration of 23-24 mg/ml andsterilized by filtration through a 0.22 μm Millipore membrane. Theintegrity of the encapsulated boron salt can be confirmed by ¹¹ B NMR at160 MHz.

The size of the liposomes can be determined by dynamic light scatteringusing methods known to those versed in the art. The encapsulatedconcentration of boron can be gaged by measuring the total boraneconcentration in each sample and then in the effluent afterultrafiltration to correct for material outside the liposomes. Theconcentration of boron can be determined by inductively coupled plasmaatomic emission spectroscopy (ICP-AES).

The therapeutic effectiveness of the borane agents of the presentinvention can be characterized by biodistribution murine studies. Allmurine biodistribution studies utilized female BALB/c mice (16-20 g),with EMT6 tumors implanted in the right flank 7-10 days prior to theexperiment. Tumor mass at the time of sacrifice was 125-350 mg.Injections of liposome emulsions (200 μl) were made in the tail vein.Prior to sacrifice, each mouse was anesthetized with halothane and bledinto heparinized syringes via cardiac puncture. The blood was thenplaced into tared cryogenic tubes. While under anesthesia, the mice wereeuthanized via cervical dislocation. The tumor, liver, and spleen weredissected and also placed in tared cryogenic tubes. Blood and tissueswere stored frozen until analyzed.

Biodistributions are plotted as boron concentration in micrograms ofboron per gram of tissue on the Y axis and time in hours on the X axis.In general, four tissues are analyzed: tumor and blood because of theirtherapeutic interest, and liver and spleen because these tissues areknown to competitively bind liposomes in vivo.

FIG. 2 shows the biodistributions of Na₂ B₁₀ H₁₀ and Na₂ B₁₂ H₁₁ SH inBALB/c mice bearing EMT6 tumors. This figure shows that both of thesecompounds are non-therapeutic as the concentration of the boron activein the tumor is initially fairly low and drops off rather rapidly.

It is believed that the borane compounds of the present inventionincluding those which are derivatives of B₁₀ H₁₀ ²⁻ are more reactivethan the prior art Na₂ B₁₀ H₁₀ because of intraceIlular protein binding.Thus, the present invention includes a method of treating tumorsincluding the step of administering a borane compound which is capableof being nucleophilically attacked by an intracellular protein in vivoand subjecting the tumor to thermal neutrons. See FIG. 3 which shows thereaction of B₁₀ H₉ NCO²⁻ with intracellular protein.

With reference to FIG. 4, one can readily see that the liposomal Na₂ B₁₀H₉ NCO derivative is far superior than the parent Na₂ B₁₀ H₁₀ moiety.The tumor uptake with Na₂ B₁₀ H₁₀ begins at 10 μg/g; whereas, theliposomal Na₂ B₁₀ H₉ NCO begins around 20. Thus, with the latter moiety,the tumor is taking up more boron by a factor of 2. Also, the drop offis not as rapid and therefore adding the NCO functional group makes theboron agent more active. The B₁₀ H₁₀ ²⁻ ion demonstrated abiodistribution characterized by a rapid clearance of the boron compoundfrom all tissues, including the tumor.

The biodistribution of B₁₀ H₉ NCO²⁻ demonstrated an initial tumor boronconcentration of approximately 20 micrograms boron per gram of tissue.This concentration remains relatively stable over a 30 hour time period.

A schematic of certain preferable of the B₁₀ H₁₀ ²⁻ derivativesaccording to the present invention is shown in FIG. 5. The preparationof these derivatives is generally described in Shelly, Hawthorne, andKnobler, Inorg. Chem. 31, 2889 (1992), which is hereby incorporated byreference.

The XB₁₀ H₉ L species of the present invention can be prepared from[2-B₁₀ H₉ CO]¹⁻ which itself is prepared by a novel reaction which isanother aspect of the present invention. It has been found that thereaction of oxalyl chloride (COCl)₂ with [B₁₀ H₁₀ ]²⁻ proceeds rapidlyand essentially quantitatively at room temperature with the evolution ofcarbon monoxide to produce the carbonyl derivative [2-B₁₀ H₉ CO]⁻ shownin Scheme I below. Preferably, the B₁₀ H₁₀ ²⁻ may take the form of [Ph₃PMe]B₁₀ H₁₀. ##STR1##

Scheme 1. Transformations of the B₁₀ H₉ CO⁻⁻ ion.

The cleanest reaction is observed employing [Ph₃ PMe][B₁₀ H₁₀ ] in CH₂Cl₂, but the reaction can be used with several salts of [B₁₀ H₁₀ ]²⁻including preferably cesium, tetramethylammonium and triethylammoniumand with other solvents such as acetonitrile and tetrahydrofuran. Thereaction product exhibits a strong infrared peak at 2129 cm¹⁻. The ¹¹B{¹ H} spectrum of the reaction mixture in CH₂ Cl₂ exhibits sevensignals, consistent with equatorial substitution, and shows only tracesof by-products. The substituted boron is indicated by a high fieldresonance at -43.8 ppm (relative to BF₃ ·Et₂ O) which is a singlet inthe proton coupled spectrum. [Ph₃ PMe][2-B₁₀ H₉ CO] may be isolated as alight tan solid in 85% yield, or the reaction mixture may be useddirectly in further reactions. The structure of [2-B₁₀ H₉ CO]⁻ has beendetermined by X-ray crystallography and shows a linear B-C-O array witha C-O distance of 1.13 A.

The utility of the [2-B₁₀ H₉ CO]⁻ anion results from its ease ofconversion to a variety of other species useful for BNCT as depictedScheme I above and FIG. 5. Although [2-B₁₀ H₉ CO]⁻ is relativelyinsensitive to moisture in the solid state or in solution, it is easilyhydrated in aqueous solvent mixtures to form [2-B₁₀ H₉ CO₂ H]²⁻. [Ph₃PMe]₂ [2-B₁₀ H₉ CO₂ H] has been confirmed by crystallographic analysis.

The reaction of [2-B₁₀ H₉ CO]⁻ with an amine (RNH₂) results in theformation of an amide ([2-B₁₀ H₉ CONHR]²⁻) as long as an excess of theamine is present to absorb the acid formed by the reaction. R is alipophilic alkyl, preferably selected from the group consisting ofethyl, propyl and hexyl. Esters are produced by the combination of[2-B₁₀ H₉ CO]⁻ with alcohols; in this case, the reaction is slow orincomplete unless an auxiliary base such as triethylamine is added toabsorb the acid formed.

A very useful transformation of [2-B₁₀ H₉ CO]⁻ occurs from its reactionwith azide ion in acetonitrile. The carbonyl undergoes a Curtius-typerearrangement to form the isocyanate [2-B₁₀ H₉ NCO]²⁻, indicated by aninfrared absorption at 2304 cm⁻¹. This ion, whose structure has beendetermined by X-ray crystallography, is stable in neutral aqueoussolution but is rapidly hydrolyzed in acidic media. The [2-B₁₀ H₉ CO]⁻ion therefore offers a high-yield route to the production of manyboron-rich species for boron neutron capture therapy. Detailed protocolsof preparing certain of these species is as follows:

[Ph₃ PMe][closo-2-B₁₀ H₉ CO] ([Ph₃ PMe]·1) A mixture of [Ph₃ PMe]₂[closo-B₁₀ H₁₀ ] (6.73 g, mmol), which is readily available from (Et₃NH)₂ B₁₀ H₁₀, in 125 mL of dry CH₂ Cl₂ was chilled in an ice bath withstirring. A solution of (COCl)₂ in CH₂ Cl₂ (5.1 mL, 10.2 mmol) was addedvia syringe, and the mixture was stirred at 0° C. for 30 min. Thesolution as allowed to warm to room temperature and stirred anadditional 30 min. The volume of the solution was reduced to ˜15 mL bymechanical vacuum. The resulting solution was passed through a 2.5×30 cmcolumn of silica gel, eluting with CH₂ Cl₂, and the effluent wasevaporated in vacuo. The residue was recrystallized from CH₂ Cl₂ /Et₂ Oto yield 3.59 g (8.5 mmol, 85%) of light tan [Ph₃ PMe₃ ]·1. Anal. Calcdfor C₂₀ H₂₇ B₁₀ OP: C, 56.85; H, 6.44; B, 25.59. Found: C, 57.01; H,6.30; B, 25.50. IR (cm⁻¹, KBr disk): 2519 (s), 2501 (s), 2129 (s). ¹¹B{¹ H} NMR (ppm, CH₂ Cl₂, relative areas in parentheses): 6.4 (1), 6.0(1), -17.9 (1), -25.9 (2), -28.4 (2), -28.9 (2), -43.8 (1). The -43.8ppm resonance was a singlet in the proton-coupled spectrum.

[Ph₃ PMe]₂ [closo-2-B₁₀ H₉ CO₂ H] ([Ph₃ PMe]₂ ·2). A mixture of [Ph₃PMe]₂ [closo-2-B₁₀ H₁₀ ] (5.52 g, 8.2 mmol) in 100 mL of CH₂ Cl₂ wasallowed to react with 4.2 mL of (COCl)₂ as described above. After vacuumremoval of the solvent, the residue was dissolved in 200 mL of hotacetone, and the solution was stirred with 3 g of activated charcoal.The mixture was filtered, and 150 mL water was added to the flitrate.The solution was neutralized by addition of 0.5N NaOH, and acetone wasallowed to evaporate from the mixture at room temperature. The resultingcrystals were filtered off and dried to give 4.21 g of [Ph₃ PMe]₂ ·2(5.9 mmol, 72%). Anal. Calcd for C₃₉ H₄₆ B₁₀ O₂ P₂ : C, 65.35; H, 6.47;B, 15.08. Found: C, 65.18; H, 6.43; B, 15.20. IR (cm⁻¹, KBr disk): 2458(s), 1694 (m), 1256 (m). ¹¹ B{¹ H} NMR (ppm, CH₂ Cl₂, relative areas inparentheses): -0.5 (1), -1.3 (1), -25.5 (1), -28.6 (3), -29.6 (4).

[Et₃ NH]₂ [closo-2-B₁₀ H₉ NCO] ([Et₃ NH]₂ ·3). A solution of [Et₃ NH]₂[closo-2-B₁₀ H₁₀ ] (3.22 g, 10 mmol) in 150 mL of MeCN was allowed toreact with 5 mL of (COCl)₂ as described above. Solid NaN₃ (1.4 g, 21mmol) was added, and the mixture was stirred overnight. The mixture wasthen filtered, 200 mL ether was added, and the solution was chilled to-20 ° C. overnight. The precipitate was filtered off and dried undervacuum, yielding 2.73 g of [Et₃ NH]₂ ·3 (7.5 mmol, 75%). Analytical andcrystallographic samples of [Et₃ NH]₂ ·3 were purified further byrecrystallization from acetone/pentane. Anal. Calcd for C₁₃ H₄₁ B₁₀ N₃O: C, 42.94; H, 11.37; N, I 1.56; B, 29.73. Found: C, 42.81; H, 11.18;N, 11.71; B, 29.50. IR (cm⁻¹, KBr disk): 2538 (s), 2505 (s), 2475 (s),1013 (m), 967 (m), 597 (w), 577 (w). ¹¹ B{¹ H} NMR (ppm, MeCN, relativeareas in parentheses): -2.3 (2), -16.8 (1), -25.2 (2), -25.6 (2), -28.3(2), -31.6 (1). The resonance at -16.8 ppm was a singlet in theproton-coupled spectrum.

The synthesis of [B₁₀ H₉ CO]⁻ is described above. The synthesis may alsobe performed starting with: Cs₂ [B₁₀ H₁₀ ], [Et₃ NH]₂ [B₁₀ H₁₀ ],[(CH₃)₄ N]₂ [B₁₀ H₁₀ ], or [Ph₃ PCH₃ ]₂ [B₁₀ H₁₀ ] in acetonitrile; or[Et₃ NH]₂ [B₁₀ H₁₀ ]; [(CH₃)₄ N]₂ [B₁₀ H₁₀ ], or [Ph₃ PCH₃ ]₂ [B₁₀ H₁₀ ]in methylene chloride. The synthesis of [B₁₀ H₉ NCO]²⁻ may be performedwith any of the above cations but has been found to proceed smoothlyonly in acetonitrile.

To synthesize [B₁₀ H₉ CO₂ C₂ H₅ ]²⁻ and [B₁₀ H₉ CO₂ CH₃ ]²⁻, [Et₃NH][B₁₀ H₉ CO] (2.5 mmol) was stirred in 50 ml of the appropriatealcohol in the presence of 0.5 mL Et₃ N for 30 minutes. The alcohol wasremoved in vacuo and the residue was recrystallized fromacetonitrile/ether. [Et₃ NH]₂ [B₁₀ H₉ CO₂ C₂ H₅ ] (in acetonitrile) ¹¹B{¹ H} 0.8, -0.4, -23.4, -27.2; [Et₃ NH]₂ [B₁₀ H₉ CO₂ CH₃ ] (inacetonitrile) ¹¹ B{¹ H} 0.8, -0.4, -23.9, -27.5.

To synthesize [B₁₀ H₉ CONHC₃ H₇ ]²⁻, [Et₃ NH][B₁₀ H₉ CO] (2.5 mmol) wasstirred in 50 mL of propylamine for 30 minutes. The propylamine wasremoved in vacuo and the residue was dissolved in ethanol containing[(CH₃)₄ N]Cl and precipitated with ether. [(CH₃)₄ N]₂ [B₁₀ H₉ CONHC₃ H₇] (in acetonitrile) ¹¹ B{¹ H} 0.3, -24.7, -26.7, -27.3.

As discussed above, one aspect of the present invention is the use inBNCT of borane moleties capable of undergoing nucleolahilic attack. Suchcompounds possess the ability to react with intracellular proteinmoleties. For example, the B₁₀ H₁₀ ²⁻ anion is known to undergooxidative coupling to produce the B₂₀ H₁₈ ²⁻ species. Compounds capableof intracellular binding include derivatives of the monocarbonylsubstituted B₁₀ H₁₀ ²⁻ ion discussed above, substituted derivatives ofB₂₀ H₁₈ ²⁻, such as an amine derivative discussed below, reduced B₂₀ H₁₈²⁻ derivatives, which may be oxidized intracellularly to produce thecorresponding reactive B₂₀ H₁₈ ²⁻ derivative, carbonyl substituted(acylium ion analogs), of B₁₀ H₁₀ ²⁻ and B₂₀ H₁₈ ⁴⁻ capable of proteinNH₂ reactions.

Salts of B₂₀ H₁₇ NH₃ ³⁻ have been shown to be particularly useful forBNCT, both free and encapsulated by liposomes. FIG. 6 shows thetetramethyl ammonium salt of B₂₀ H₁₇ NH₃ ³⁻.

To prepare another salt of B₂₀ H₁₇ NH₃ ³⁻, Na₃ B₂₀ H₁₇ NH₃,approximately 175 mL of liquid ammonia is condensed in a flaskcontaining 3 mmol of dry Na₂ (n-B₂₀ H₁₈) and cooled by both a dryice/acetone bath and condenser. An 18% suspension of sodium acetylide inxylene/light mineral oil (2.0 mL, 8 mmol of NaC₂ H) is added dropwisevia syringe. The dry ice/acetone bath is removed from the base of thereaction flask and the condenser is maintained for 3 hours. The ammoniais allowed to evaporate under a nitrogen atmosphere. The remainingsolvent is removed in vacuo. Absolute ethanol (50 mL) is added and theresulting solution filtered. The product is precipitated using asaturated solution of (CH₃)₄ NCl in absolute ethanol. The solid isfiltered and recrystallized from water/ethanol in 70% yield. The cationis exchanged to Na using standard methods. 160 MHz ¹¹ B NMR (CHCl₃, δreferenced to external BF₃ ·OEt₂): 9.4 (s, 1 B), 3.0 (d, 1 B), -1.4 (d,1 B), -7.1 (d, 1 B), -14.9 (s, 1 B), -24.8 (d), -26.2 (d), -29.0 (d),-31.0 (d).

FIG. 7 shows the biodistribution characterization of Na₃ B₂₀ H₁₇ NH₃encapsulated in the 1:1 DSPC/cholesterol liposome discussed above inBALB/c mice bearing EMT6 tumors. Quite significantly, the tumor boronconcentration increases over a period of approximately 30 hours and thenslowly decreases, resulting in a final tumor boron concentration whichis still 94% of the initial tumor boron concentration. All other tissuesclear steadily over the 40 hour time period. The low blood boronconcentration at 48 hours yields a tumor to blood boron ratio of 5.3.The final tumor boron concentration of 25.4 micrograms boron per gramtissue is approximately equal to the initial tumor boron concentrationand is well within therapeutic levels. It is believed that the speciesB₂₀ H₁₇ NH₃ ³⁻ is oxidized within the cell according to the followingScheme 2 and the resulting ion is very reactive to nucleophiles, forexample, the terminal amine groups in the tumor cell. The singlenegative charge of the resulting B₂₀ H₁₇ NH₃ ⁻ gives this speciesenhanced electrophilicity compared to B₂₀ H₁₈ ²⁻ ions and improvedintracellular bonding. ##STR2##

In general, in addition to NH₃, other reduced, substituted B₂₀ H₁₈ ²⁻derivatives are believed to be subject to intracellular oxidation afterliposomal delivery according to Scheme 2. As shown in FIG. 8, the B₂₀H₁₇ L¹⁻ species is particularly reactive with nucleophiles, e.g.,intracellular proteins in tumor cells. Thus, the present inventionincludes the more reactive borane compounds X_(y) B₂₀ H₁₇ L as discussedabove and the corresponding liposome encapsulated compounds where y is 1to 4. Methods of preparing certain of the X_(y) B₂₀ H₁₇ L derivativesare as follows:

The amine derivatives of [B₂₀ H₁₈ ]²⁻, [B₂₀ H₁₇ NH₂ R]³⁻, aresynthesized in the following manner:

Dry Na₂ [n-B₂₀ H₁₈ ] (3 mmol) is dissolved in the desired amine (30 mL)under and atmosphere of nitrogen. Sodium acetylide, NaC₂ H (2.0 mL, 18%in a suspension of xylene/light mineral oil) is added to the solutionvia syringe. The mixture is allowed to stir for approximately one day atroom temperature. The amine is removed in vacuo and absolute ethanol (50mL) is added to the residue. The product is precipitated as thetetramethylammonium salt by the addition of a saturated solution of(CH₃)₄ NCl in absolute ethanol. Recrystallization of the precipitatefrom water/ethanol affords the desired product in approximately 70%yield. Benzylamine, (B₂₀ H₁₇ NH₂ CH₂ C₆ H₅)³⁻ :¹¹ B{¹ H} 10.0, 2.7,-2.0, -8.2, -13.0, -26.7, -29.6; Octylamine, (B₂₀ H₁₇ NH₂ (CH₂)₇ CH₃)³⁻:¹¹ B{¹ H} 9.0, 3.0, -1.0, -6.5, -12.5, -25.9, -28.8; andEthylenediamine, (B₂₀ H₁₇ NH₂ CH₂ CH₂ NH₂)³⁻ : ¹¹ B{¹ H} 9.0, 2.8, -0.9,-3.9, -6.3, -12.4, -28.1.

To prepare the oxidized amine species [B₂₀ H₁₇ NH₃ ]¹⁻, to a solution of0.7 g of [(CH₃)₄ N][B₂₀ H₁₇ NH₃ ] in 40 mL distilled water at 0° C. wasadded slowly 20 mL of a 0.35M ferric chloride solution (FeCl₃ ·6H₂ O).The reaction mixture was stirred for approximately one day. The product,which precipitated from solution, was filtered and dried.

To synthesize [B₂₀ H₁₇ CN]⁴⁻, [Et₃ NH]₂ [n-B₂₀ H₁₈ ] (0.54 g, 1.2 mmol)was dissolved in 15 mL distilled acetonitrile. While under a nitrogenatmosphere, 0.86 g (5.5 mmol) Et₄ NCN was added. The solution wasrefluxed for approximately 24 hours. Cyano derivative, [B₂₀ H₁₇ CN]⁴⁻ :¹¹ B{¹ H} 10.4, 0.8, -2.5, -4.5, -12.9, -23.2, -26.2, -27.1.

To synthesize [B₂₀ H₁₇ SH]⁴⁻, dry Na₂ [n-B₂₀ H₁₈ ] (2.5 mmol) isdissolved in a combination of distilled acetonitrile (50 mL) anddistilled ether (20 mL). The solution is transferred via cannula to aflask containing anhydrous NaSH (0.31 g, 5.5 mmol). The solution isrefluxed for approximately one week. The solvent was then removed invacuo. Absolute ethanol (50 mL) was added which precipitated the productas the tetramethylammonium salt by adding a saturated solution of (CH₃)₄NCl in absolute ethanol. Thiol derivative, [B₂₀ H₁₇ SH]⁴⁻ : ¹¹ B{¹ H}4.1, 1.6, -0.7, -23.2, -24.6, -27.2.

The previous example and FIG. 7 involved the encapsulation of Na₃ B₂₀H₁₇ NH₃ in a liposome having an approximately 110 nm average diameter.It has also been found that larger liposomes having active boranecompounds encapsulated therein, or perhaps aggregates of similarliposomes, having a mean diameter of from 100 to 200 and preferably lessthan 250 nm also show therapeutic value. Reference here is made to FIG.9 where the boron concentration in the tumor begins at 30 μg/g andincreases over time to 60 μg/g.

Reference to therapeutic or therapeutic value or therapeutic amount orconcentration is meant to refer to that amount of the borane specieswhen disposed within a tumor cell and exposed to thermal neutronsresults in the killing of tumor cells. This concentration range assumesuse of 95% enriched ¹⁰ B. If natural abundance or lower percentageenriched ¹⁰ B is used, a higher concentration will be required as can bedetermined by routine experimentation.

In another aspect of the present invention, it has been found that freeNa₃ B₂₀ H₁₇ NH₃ also shows enhanced boron activity and tumorspecificity. By free, it is meant that this compound is not encapsulatedin a liposome. It is simply present when administered in a buffer suchas phosphate buffered lactose or saline, and administered by injection.As shown in FIG. 10, this compound may provide therapeutic delivery byinfusion over a long period of time thereby allowing accumulation withinthe tumor. It should be appreciated, that in general, other prior artfree boron derivatives injected at comparable doses have not showninitial accumulation above 2 ppm; whereas, as shown in FIG. 10, theinitial distribution in the tumor is about 8 ppm.

A comparison of the effectiveness of liposomal encapsulated boranes isshown in FIG. 11. The liposomal encapsulated boranes were prepared andthe biodistribution measured as discussed above. It is shown that theB₂₀ H₁₇ NH₃ ³ - species is far superior.

Another aspect of the present invention involves the embedment oflipophilic boron species in the phospholipid bilayer of the liposome. Itis known that unilamellar liposomes of the type illustrated above asboron delivery vehicles preferentially deliver their contents to tumorcells in animals and humans in such a manner that tumor levels ofeffector molecules are 5-10 times that of normal tissue, includingblood. According to this other aspect of the present invention it hasbeen found that embedding certain carborane species into the liposomebilayer increases the tumor specificity of the liposome. By embeddingthe carborane tumor treating agents in the lipophilic bilayer membrane,the total amount of boron to be delivered by the liposome having aborane tumor treating agent encapsulated therein can also be increased.

This aspect of the present invention is directed to liposomes preferablyunilamellar having at least one encapsulating bilayer and an internalspace defined by the encapsulating bilayer where a carborane is embeddedin the bilayer. This aspect of the present invention also involves theuse of such carborane embedded liposomes for tumor treatment in BNCT, toincrease the specificity of the liposome and to encapsulate therapeuticmoleties such as the borane species discussed above and others known tothose of skill in the art.

Liposomes with boron compounds embedded in the bilayer are prepared byhydrating the phospholipids in the presence of the boron compound to beembedded. Specifically, liposome emulsions were prepared by probesonication of a dried film composed of a lipophilic boron species, inthe desired amount, and equimolar amounts of the phospholipid andcholesterol with the hydrating solution (typically 250-300 mM in theboron-containing salt) at 65° C. for 15-30 minutes. The dried film isprepared by dissolving the lipophilic boron compound and thecholesterol:DSPC mixture in chloroform and removing the solvent invacuo. The vesicles were homogenized by sonication and were separatedfrom the remaining free borane salt by eluting through a column ofSephadex G-25 (medium) with isotonic phosphate-buffered saline orlactose. Liposomal preparations were diluted with the appropriate bufferto a lipid concentration of 5-30 mg/mL and sterilized by filtrationthrough a 0.22 μm Millipore membrane.

The amount of boron embedded in the bilayer is ideally controlled by theamount of boron compound added to the DSPC:Cholesterol lipid mixture.Assuming the boron compound is not water soluble, all of the boroncompound should be embedded, although this is not always the case. Thequantity of boron embedded in a liposome is determined by ICP-AESanalysis when no boron containing compound is encapsulated.

In general, to increase liposome specificity and for BNCT purposes,there is embedded in the bilayers preferably from 0.5 to 10 percent byweight and more preferably from 1 to 5 percent. Preferably, the bilayercomprises a phospholipid, such as DSPC and cholesterol, where the molepercent ratio of DSPC to cholesterol ranges from 1:1 to 3:1 and ispreferably 1:1.

The carborane embedded within the liposome according to the presentinvention is generally selected from the group consisting of (a), (b),(c), (d), (e), (f) and (g) and mixtures thereof as follows:

(a) A carborane of the formula: ##STR3##

R is a lipophilic alkyl group, preferably --(CH₂)_(x) CH₃ where x rangesfrom 11 to 19, more preferably R is selected from n-C₁₆ H₃₃ and n-C₁₈H₃₇. A long carbon chain, for example an R group of 16 carbon atoms,increases the stability of the bilayer because of the similaritiesbetween this compound and the phospholipid DSPC as evidenced by thepolar head group and the long lipophilic side chains.

(b) A carborane of the formula: ##STR4##

With reference to FIG. 12, the double tailed carborane species A embedsin the bilayer better than the single tailed carborane species (a)discussed above.

The embedment of a boronated species characterized by a polar head groupand two fatty acid tails as shown in FIG. 12 in the liposome bilayerstabilizes the bilayer membrane because it more closely mimics thestructure of the phospholipid utilized. The synthesis of the two tailedcompound is based on that described in Li, Ji.; Logan, C. F.; Jones, M.Jr.; Inorg. Chem. (1991), 30, 4866. Iodination of orthocarbonanefollowed by alkylation with a suitable Grignard reagent produces adialkylated closo-carborane species. Degradation of this compoundproduces a species characterized by a polar head group, thenido-carborane, and two fatty acid tails, the two C₁₈ H₃₇ chains.

(c) A metallocarborane of the formula: ##STR5##

M is a transition metal preferably selected from Fe^(III), Cr^(III) andCo^(III). The cobalt derivative and its synthetic sequence is shown inFIG. 13.

(d) A carborane of the formula: ##STR6##

R₁ and R₂ for carborane species (b), (c) and (d) are the same ordifferent and are selected from the lipophilic alkyl groups R discussedabove with respect to carborane species (a), or R₁ or R₂ is H and theother of R₁ or R₂ is such a lipophilic alkyl group.

(e) A carborane of the formula: ##STR7## where n is preferably 12, 14,16 or 18.

The cation X for each of (a)-(e) is preferably selected from the groupconsisting of the alkali metals and tetra alkyl ammoniums discussedabove with respect to X_(y) B₂₀ H₁₇ L.

(f) A closo-carborane of the formula: ##STR8##

R is as defined above. This is prepared, for R=n-C₁₆ H₃₃, as discussedbelow.

(g) A closo-carborane of the formula: ##STR9##

R₁ and R₂ are defined above, preferably --(CH₂)_(x) CH₃, where x rangesfrom 5 to 19. This is prepared for R=n-C₁₆ H₃₃, as discussed below.

As discussed above, the specificity of a unilamellar liposome can beincreased by embedding a carborane within the liposome bilayer.Preferably the carborane is selected from species (a)-(g) above ormixtures thereof.

Protocols for preparing certain of these carborane species is asfollows:

Preparation of the Dicarbollide Dianion, NaK-(3)-1,2-B₉ C₂ H₁₀ R. Asolution of 5.0 g (25.9 mmoles) of NaK-1,2-B₉ C₂ H₁₁ R which is readilyavailable in 75 ml of tetrahydrofuran was added slowly to a stirringsuspension of sodium hydride, 1.51 g (63 mmoles, 2.70 g of a 56%dispersion in mineral oil which had been washed twice with 30 ml oftetrahydrofuran), in 90 ml of the same solvent. The reaction mixture wasstirred at reflux temperature under nitrogen for 3 hr. Stirring was thenstopped and the reaction mixture allowed to cool to room temperature.When the excess sodium hydride had settled, the clear tetrahydrofuransolution of NaK-1,2C₂ B₉ H₁₀ R was collected.

Preparation of the (3)-1,2-Dicarbollylcobalt (III) Derivatives,Cs[(3)-1,2-B₉ C₂ H₁₀ R]₂ Co. To a stirred suspension of 1.20 g (9.2mmoles) of anhydrous cobaltous chloride in dry tetrahydrofuran (50 ml)was added under nitrogen a tetrahydrofuran solution of NaK-(3)-1,2-B₉ C₂H₁₀ R (5.2 mmoles) which is readily available as noted above. Theresulting black mixture was refluxed for 2 hr under nitrogen, cooled,and filtered to remove the cobalt metal and sodium chloride. After theremoval of the solvent in vacuo, the residue was extracted with hotwater, the resulting aqueous solution filtered, and the flitrate treatedwith cesium chloride.

To prepare 9,12-[CH₃ (CH₂)₁₇ ]₂ -1,2-C₂ B₁₀ H₁₀, a mixture of 2.31 gmagnesium (95 mmol) and 31.7 g CH₃ (CH₂)₁₇ Br was refluxed in 200 mL THFfor 3 h. The Grignard reagent was slowly added to a suspension of 9,12-C₂ B₁₀ H₁₀ I₂ (5.01 g, 12.7 mmol), Pd[P(C₅ H₅)₃ ]₂ Cl₂ (227 mg), andCul (65 mg) in 50 mL THF at 0° C., and then brought to reflux for 90 h.After cooling the solvent was removed in vacuo. The residue was digestedin 250 mL ether and washed with 50 mL water, 50 mL 1 N HCl, and 50 mLwater. The ether solution was dried over magnesium sulfate and thesolvent removed on a rotary evaporator. The crude product was purifiedby chromatography on silica, eluting with hexane, to yield 2.4 g9,12-[CH₃ (CH₂)₁₇ ]₂ -1,2-C₂ B₁₀ H₁₀ (30%). 160 MHz ¹¹ B NMR (CHCl₃, δreferenced to external BF₃ ·OEt₂): 8.7 (s, 2 B), -8.8 (d, 4B), -14.8 (d,4 B), -16.0 (d, 2 B).

To prepare K⁺ [5,6-[CH₃ (CH₂)₁₇ ]₂ -7,8-C₂ ₉ H₁₀ ], 1.04 g 9,12-[CH₃(CH₂)₁₇ ]₂ -1,2-C₂ B₁₀ H₁₀ (1.6 mmol) was added to a solution of 1 g KOHin 60 mL ethanol and refluxed for 24 h. The solution was cooled,saturated with carbon dioxide, filtered, and the solvent was removedunder vacuum. The residue was recrystallized from benzene to yield 1.02g (94%) K⁺ [5,6-[CH₃ (CH₂)₁₇ ]₂ -7,8-C₂ B₉ H₁₀ ]⁻. 160 MHz ¹¹ B NMR(CHCl₃, δ referenced to external BF₃ ·OEt₂): -5.0 (s, 2B), -11.6 (d,2B), -18.9 (d, 1 B), -21.0 (d, 2 B), -29.5 (d, 1 B), -35.6 (d, 1 B).

To prepare 1-CH₃ (CH₂)₁₅ -1,2-C₂ B₁₀ H₁₁, a mixture of 12.27 g B₁₀ H₁₄(100 mmol), 150 mL benzene, and 42 mL acetonitrile were refluxedovernight. Octadecyne (25.00 g, 100 mmol) was added dropwise to therefluxing solution and the solution refluxed an additional 36 hours.After cooling the solvent was removed in vacuo. Dissolved residue in 1:1ether:pentane and extracted 5×100 mL with 1N NaOH. The organic fractionwas collected, dried over Mg₂ SO₄, and the solvent removed on a rotaryevaporator. The crude product was purified by chromatography on silica,eluting with pentane. The product was isolated in 30% yield. 160 MHz ¹¹B NMR (pentane, δ referenced to external BF₃ ·OEt₂): -1.7 (d, 1 B), -5.1(d, 1 B), -8.6 (d, 2 B), -10.7 (d, 2 B), -11.5 (d, 2 B), -12.4 (d, 2 B).

To prepare K⁺ [7-[CH₃ (CH₂)₁₅ ]-7,8-C₂ B₉ H₁₀ ]⁻, 5.19 g 1-CH₃ (CH₂)₁₅-1,2-C₂ B₁₀ H₁₁ (14 mmol) was added to a solution of 1.8 g KOH in 100 mLethanol and refluxed for 24 h. The solution was cooled, saturated withcarbon dioxide, filtered, and the solvent was removed under vacuum, Theresidue was extracted using benzene in a Soxhlet extraction apparatusand the product was isolated in 90% yield. 160 MHz ¹¹ B NMR (C₆ H₆, δreferenced to external RF₃ ·OEt₂): -9.4 (d, 2 B), -12.4 (d, 1 B), -15.8(d, 1 B), -16.5 (d, 2 B), -20.7 (d, 1 B), -31.7 (d, 1 B), -35.6 (d, 1B).

With reference to FIG. 13, when the potassium carborane derivativespecies 13A is embedded in the bilayer, the resulting liposome is anegative liposome which leads to a more selective liposome and betterbiodistribution.

Biodistribution studies were performed on carborane embedded liposomes.With reference to FIG. 14, an isotonic buffer, i.e., phosphate bufferedlactose is encapsulated in the internal space of liposomes doped withK[CH₃ (CH₂)₁₅ C₂ B₉ H₁₁ ], an example of carborane species (a). The 6hour tumor value is approximately 18 micrograms of boron per gram oftumor. This boron concentration is maintained over a period of 30 hoursand then begins to decrease, resulting in a final tumor boronconcentration of 9.5 micrograms boron per gram of tumor and a finaltumor to blood boron ratio of 6.6.

With reference to FIG. 15, a hypertonic buffer (375 mM NaCl/10% HEPESbuffer) is encapsulated in the aqueous core (or internal space) ofliposomes doped with K(C₂ B₉ H₁₁)(CH₂)₁₅ CH₃. The hypertonic buffer moreclosely mimics the actual osmotic stress on the liposomes resulting fromencapsulated borane solutions. The mean diameter of the liposomes (99nm) is within the normal range of liposomes desired for BNCT. Theinjected dose of these liposomes (6.3 mg/kg body weight) is slightlyhigher than the isotonic buffer example of FIG. 14. The 6 hour tumorvalue is approximately 22 μg boron/g tumor. This boron concentrationincreases for approximately 12 hours and then decreases over theremaining time period, resulting in a final tumor boron concentration of25.2 μg boron/g tumor and a final tumor to blood boron ratio of 8.4,both of which are well within therapeutic values (20 μg boron/g tumorand a tumor to blood boron ratio of 3).

In both FIG. 14 and FIG. 15, the liver and spleen boron concentrationsare significantly lower than observed in other in vivo biodistributions.

Another aspect of the present invention involves treating tumors byadministering to the patient a therapeutically effective amount of aunilamellar liposome having at least one encapsulating bilayer and aninternal space defined by the encapsulating bilayer wherein a carboraneof species (a)-(e) or mixtures thereof is embedded in the bilayer. Inanother aspect of the present invention, the carborane embeddedliposomes as discussed above include a borane compound within theinternal space of the liposome. This will increase the amount of ¹⁰ Bavailable for BNCT when a borane encapsulated liposome is used. Theencapsulated borane compounds can be selected from the borane speciesdiscussed above, namely X_(y) B₂₀ H₁₇ L and X_(s) B₁₀ H₉ L, or otherdrugs to be delivered to the tumor. Most preferably, the borane isselected from the group consisting of N_(y) B₂₀ H₁₇ NH₃ where Y is 1 or3, and Na₂ B₁₀ H₉ NCO. The method of treating tumors according to thisaspect of the present invention comprises administering the carboraneembedded liposomes discussed above and exposing the tumors to thermalneutrons.

In all methods according to the present invention, the compounds areadministered by i.v. injection with tumor concentration monitored bysacrifice at selected time points. Preferably, the initial boron tumorconcentration is at least 10 μg/g tumor, preferably from 20 to 30, butthe highest possible concentration is desired. If the desired borontumor concentration is not up to therapeutic concentration, thepharmaceutical suspension containing the boron agent can be infused overa long period of time or given by multiple injection.

Although this invention has been described with reference to particularapplications, the principals involved are susceptible to otherapplications which will be apparent to those skilled in the art. Theinvention is accordingly to be limited only by the scope of the appendedclaims.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:

What is claimed:
 1. The compound Na_(y) B₂₀ H₁₇ NH₃, where y is 1 or 3.2. A water soluble salt of B₂₀ H₁₇ NHR₁ R₂ ^(-z), where R₁ and R₂ arethe same or different and are selected from the group consisting ofhydrogen, benzyl, alkyl and diamine, and z is 1 or
 3. 3. The salt ofclaim 2 wherein the salt comprises a cation which is selected from thegroup consisting of alkali metals and tetra alkyl ammonium wherein thealkyl is any alkyl where the water solubility of the salt is retained.4. The salt of claim 3 wherein the cation is tetramethylammonium.
 5. Thesalt of claim 2 wherein R₁ and R₂ are selected from the group consistingof ethyl and diamine.